BIMS 808 General and Molecular Genetics

1. BIMS 808General and Molecular Genetics Instructor is not an expert on DNA repair, so strongly advised to check out the references for more in depth treatment. Most of the figures to be presented come from the special issue of Cell Research, so when not credited that is where they come from. Friedberg text is THE comprehensive text on the subject and is reasonably current with latest edition in 2006. Unfortunately, the newest copy at any UVA library is 1995.Instructor is not an expert on DNA repair, so strongly advised to check out the references for more in depth treatment. Most of the figures to be presented come from the special issue of Cell Research, so when not credited that is where they come from. Friedberg text is THE comprehensive text on the subject and is reasonably current with latest edition in 2006. Unfortunately, the newest copy at any UVA library is 1995.

2. Mutation, while not the driving force of natural selection, is fundamental to its operation by providing the necessary variation. DNA repair could have been predicted but was not immediately obvious to those who first determined that DNA was the material of inheritance.Mutation, while not the driving force of natural selection, is fundamental to its operation by providing the necessary variation. DNA repair could have been predicted but was not immediately obvious to those who first determined that DNA was the material of inheritance.

3. Some history. . . . Hugo de Vries, Principles of the theory of mutation. Science (1914) First coined the term “mutation” Idea that genetic variation arises from some unspecified “intracellular perturbation” Hermann J. Muller, Artificial transmutation of the gene, Science (1927) X-ray mutagenesis of Drosophila In a single experiment increased the known number of Drosophila mutants by 50% De Vries is also responsible for the term “gene” from his paper on pangenesis from 1889. Need to realize that at this time, the idea of a “gene” was just an abstract concept. It was not clear that genes had any physical reality. So it is important that de Vries first proposed that a mutation represented some physical change in a cell. Muller’s demonstration of inducible mutation shocked most geneticists including major figures like Thomas Hunt Morgan. It was widely anticipated that it would prove to be false but within a few years, most scientists, including Morgan, were using it as a tool to map genes.De Vries is also responsible for the term “gene” from his paper on pangenesis from 1889. Need to realize that at this time, the idea of a “gene” was just an abstract concept. It was not clear that genes had any physical reality. So it is important that de Vries first proposed that a mutation represented some physical change in a cell. Muller’s demonstration of inducible mutation shocked most geneticists including major figures like Thomas Hunt Morgan. It was widely anticipated that it would prove to be false but within a few years, most scientists, including Morgan, were using it as a tool to map genes.

4. More history. . . Delbruck’s target theory of gene inactivation—1935 UV induction of mutations--1941 Maximal induction at 265nm wavelength Avery demonstrates that genes are constructed of DNA—1944 Kelner and Dulbecco separately discover that UV induction of mutations can be counter-acted by exposure to visible light It is worth noting that Delbruck was a physicist at this time working in a physics lab in Berlin. He co-authored this paper in an obscure German language physics journal. However, the idea that one could map genes based on their target size for X-rays was prescient. Many investigators were using UV light for mutagenesis by this time. Although it was known that the frequency of mutations varied with the wavelength and that the maxima coincided with the wavelength at which DNA maximally absorbed, nobody put this together. Kelner and Dulbecco both struggled with systematic errors in their studies; Kelner because plates left near a window had fewer mutations and Dulbecco because the frequency of mutation increased the farther down you went in a stack of plates!It is worth noting that Delbruck was a physicist at this time working in a physics lab in Berlin. He co-authored this paper in an obscure German language physics journal. However, the idea that one could map genes based on their target size for X-rays was prescient. Many investigators were using UV light for mutagenesis by this time. Although it was known that the frequency of mutations varied with the wavelength and that the maxima coincided with the wavelength at which DNA maximally absorbed, nobody put this together. Kelner and Dulbecco both struggled with systematic errors in their studies; Kelner because plates left near a window had fewer mutations and Dulbecco because the frequency of mutation increased the farther down you went in a stack of plates!

5. DNA damage induced by UV light The damage that investigators were generating with UV light were primarily of two kinds: cyclobutane thymine dimers forming between adjacent thymine residues or, less commonly, 6-4 photoproducts, again between adjacent thymine residues. These lesions create kinks in the DNA that block transcription and replication and, as we shall see, serve as a way for the cell to recognize them.The damage that investigators were generating with UV light were primarily of two kinds: cyclobutane thymine dimers forming between adjacent thymine residues or, less commonly, 6-4 photoproducts, again between adjacent thymine residues. These lesions create kinks in the DNA that block transcription and replication and, as we shall see, serve as a way for the cell to recognize them.

6. Hints of the ability of living cells to recover from the lethal effects of e.g. UV began to emerge as early as the mid-1930s. Actual repair was observed by Dulbecco and Kelner independently in the 1940s when they observed the light dependent ability of cells or phage to recover from UV light-induced damage. Discovery of photolyase was entirely serendipitous. Photolyases exist in most organisms, but are a minor mechanism of repair in higher eukaryotes. They represent a prime example of repair by direct reversal. Hints of the ability of living cells to recover from the lethal effects of e.g. UV began to emerge as early as the mid-1930s. Actual repair was observed by Dulbecco and Kelner independently in the 1940s when they observed the light dependent ability of cells or phage to recover from UV light-induced damage. Discovery of photolyase was entirely serendipitous. Photolyases exist in most organisms, but are a minor mechanism of repair in higher eukaryotes. They represent a prime example of repair by direct reversal.

7. Even more history. . . 1950s An E.coli strain hypersensitive to UV isolated Liquid holding recovery independent of visible light 1960s Loss of DNA fragments in bacteria recovering from UV Multiple complementation groups of E. coli UV sensitive mutants Repair synthesis in E. coli What about mammals (like humans)? First evidence is developed that repair is genetically encoded and controlled. Liquid holding experiments suggest the need for time to effect repair and presage the existence of cell cycle checkpoints in eukaryotes. By the 1960s there is evidence for other mechanisms of repair and tools such as antibodies to thymine dimers or BUdR incorporation that can be used to assay these other methods of repair directly without having to measure mutations.First evidence is developed that repair is genetically encoded and controlled. Liquid holding experiments suggest the need for time to effect repair and presage the existence of cell cycle checkpoints in eukaryotes. By the 1960s there is evidence for other mechanisms of repair and tools such as antibodies to thymine dimers or BUdR incorporation that can be used to assay these other methods of repair directly without having to measure mutations.

8. Xeroderma pigmentosum (XP) In humans, UV damage repaired only by nucleotide excision repair (NER) Defects in NER lead to at least 3 different genetic disorders: XP Cockayne syndrome Trichothiodystrophy It’s often overlooked how early (1968) and important Jim Cleaver’s recognition of XP as a DNA repair disorder was. This one observation tied together genetics, DNA repair and cancer showing for the first time that cancer was a genetic disorder and could arise through damage to DNA. He actually learned of XP from an article in the newspaper describing the disorder from a meeting report by Henry Lynch. As we shall see, the genetics of XP are highly complex, with multiple complementation groups and two other overlapping genetic disorders.It’s often overlooked how early (1968) and important Jim Cleaver’s recognition of XP as a DNA repair disorder was. This one observation tied together genetics, DNA repair and cancer showing for the first time that cancer was a genetic disorder and could arise through damage to DNA. He actually learned of XP from an article in the newspaper describing the disorder from a meeting report by Henry Lynch. As we shall see, the genetics of XP are highly complex, with multiple complementation groups and two other overlapping genetic disorders.

9. Magnitude of the DNA repair problem The magnitude of the danger to DNA, as highlighted in the opening quote from Crick, cannot be overstated. There are numerous environmental threats such as radiation, radon, pollution, chemicals, but perhaps more dangerous are the endogenous sources of damage. Anytime you put oxygen or water together with DNA there is the potential for trouble. In a cell reactive oxygen species are constantly being formed as are result of cellular metabolism and these are a major source of possible damage.The magnitude of the danger to DNA, as highlighted in the opening quote from Crick, cannot be overstated. There are numerous environmental threats such as radiation, radon, pollution, chemicals, but perhaps more dangerous are the endogenous sources of damage. Anytime you put oxygen or water together with DNA there is the potential for trouble. In a cell reactive oxygen species are constantly being formed as are result of cellular metabolism and these are a major source of possible damage.

10. The major mechanisms of repair for nucleotide damage in mammals are via excision Base Excision Repair (BER) Repair of oxidizes, alkylated or inappropriate bases as well as abasic sites Responsible for repair of the majority of DNA damage Nucleotide Excision Repair (NER) Mechanism of DNA excision and repair synthesis that corrects damage caused by agents that create bulky DNA adducts (e.g. thymine dimers) Most versatile of excision repair mechanisms Mismatch Repair (MMR) Repairs base-base mismatches and insertion/deletion mispairings arising during DNA replication and recombination For most of our discussion, we will focus on mammals, particularly humans, because I want to tie this information together with genetic disorders, cancer and therapy. However, most organisms employ many different repair mechanisms. As a general rule, these pathways are damage specific (with some overlap). As we shall see, most operate by excising the damage and replacing it with (hopefully) the correct sequence.For most of our discussion, we will focus on mammals, particularly humans, because I want to tie this information together with genetic disorders, cancer and therapy. However, most organisms employ many different repair mechanisms. As a general rule, these pathways are damage specific (with some overlap). As we shall see, most operate by excising the damage and replacing it with (hopefully) the correct sequence.

11. Alkylation: addition of ethyl or methyl groups to any nucleophilic atom on DNA Alkylation is a frequent cause of DNA damage that is repaired by BER. Alkylation is both cytotoxic and genotoxic and many chemotherapeutic agents are alkylators. In addition to potentially creating bulky adducts that can interfere with replication, alkylation can affect base pairing. In this case, alkylation of guanine allows it to pair incorrectly with thymine and changes the DNA sequence.Alkylation is a frequent cause of DNA damage that is repaired by BER. Alkylation is both cytotoxic and genotoxic and many chemotherapeutic agents are alkylators. In addition to potentially creating bulky adducts that can interfere with replication, alkylation can affect base pairing. In this case, alkylation of guanine allows it to pair incorrectly with thymine and changes the DNA sequence.

12. As mentioned earlier in the context of photolyases, one mechanism of repair of damage is direct reversal. In this case, MGMT removes the offending alkyl group from a methylated guanine.As mentioned earlier in the context of photolyases, one mechanism of repair of damage is direct reversal. In this case, MGMT removes the offending alkyl group from a methylated guanine.

13. Basic mechanism of BER Excision of damaged base by specific DNA glycosylase Creates an abasic (AP) site AP endonuclease (APE) cleaves the AP site Generates 3’OH and 5’ deoxyribose termini DNA polymerase fills the single nucleotide gap A DNA ligase seals the nick after synthesis BER proceeds via a multi-step process. Specificity is provided through the recognition of damage which is carried out by a family of DNA glycosylases.BER proceeds via a multi-step process. Specificity is provided through the recognition of damage which is carried out by a family of DNA glycosylases.

14. DNA glycosylases confer specificity on BER Two things to notice here. First, there are a lot of different DNA glycosylases and they target different kinds of damage. Second, some of the glycosylases are bifunctional; they both excise the damaged base, and cleave the resulting AP site. Bifunctional glycosylases are invariably associated with repair of oxidized bases. In mammals, none of the glycosylases involved in processing oxidized bases is essential. Knockouts for all have been constructed and are viable. They continue to accrue damage such as 8-oxo-G, but do not display an increase in cancer. This contrasts sharply with NER and MMR. Not clear how damage is processed in these cases. Some by NER but perhaps there is more redundancy in glycosylase function than has been reported.Two things to notice here. First, there are a lot of different DNA glycosylases and they target different kinds of damage. Second, some of the glycosylases are bifunctional; they both excise the damaged base, and cleave the resulting AP site. Bifunctional glycosylases are invariably associated with repair of oxidized bases. In mammals, none of the glycosylases involved in processing oxidized bases is essential. Knockouts for all have been constructed and are viable. They continue to accrue damage such as 8-oxo-G, but do not display an increase in cancer. This contrasts sharply with NER and MMR. Not clear how damage is processed in these cases. Some by NER but perhaps there is more redundancy in glycosylase function than has been reported.

15. Unifying feature of DNA glycosylases is that they scan for DNA damage by extrahelical flipping of damaged bases Shows structure of AAG (aka MPG or ANPG) methyl adenine DNA glycosylase. Can also perform BER on other modified bases to varying degrees. Strategy of flipping out base is one generally used by enzymes that scan duplex DNA for base aberrations. All known glycosylases bind in the minor groove of DNA, kink the DNA at the site of the lesion and flip the lesion out of the DNA major groove.Shows structure of AAG (aka MPG or ANPG) methyl adenine DNA glycosylase. Can also perform BER on other modified bases to varying degrees. Strategy of flipping out base is one generally used by enzymes that scan duplex DNA for base aberrations. All known glycosylases bind in the minor groove of DNA, kink the DNA at the site of the lesion and flip the lesion out of the DNA major groove.

16. “Flipped” base must fit into a highly specific active site in the glycosylase Shows structure of AAG (aka MPG or ANPG) methyl adenine DNA glycosylase. Can also perform BER on other modified bases to varying degrees. Strategy of flipping out base is one generally used by enzymes that scan duplex DNA for base aberrations. In this image, one sees how the damaged base fits snugly within the enzyme active site pocket. The tightness of fit may explain why most DNA glycosylases are poor enzymes with low turnover rates. For bifunctional glycosylases the story may be different. There are many types of oxidative damage but very few glycosylases suggesting that there must be broader specificities.Shows structure of AAG (aka MPG or ANPG) methyl adenine DNA glycosylase. Can also perform BER on other modified bases to varying degrees. Strategy of flipping out base is one generally used by enzymes that scan duplex DNA for base aberrations. In this image, one sees how the damaged base fits snugly within the enzyme active site pocket. The tightness of fit may explain why most DNA glycosylases are poor enzymes with low turnover rates. For bifunctional glycosylases the story may be different. There are many types of oxidative damage but very few glycosylases suggesting that there must be broader specificities.

17. Why doesn’t DNA have uracil? One common type of damage is deamination. When cytosine is deaminated, it becomes uracil. While uracil is used in RNA, in DNA it’s propensity to pair with either A or G makes it potentially mutagenic. The cell would not know when a mutation had occurred. Therefore, BER acts to remove U’s when they occur in DNA. As an aside, deamination of 5-methyl-C creates thymine. Since C is the nucleotide typically methylated in eukaryotic cells, this is a significant problem because the cell cannot readily discern which base is “correct”. One common type of damage is deamination. When cytosine is deaminated, it becomes uracil. While uracil is used in RNA, in DNA it’s propensity to pair with either A or G makes it potentially mutagenic. The cell would not know when a mutation had occurred. Therefore, BER acts to remove U’s when they occur in DNA. As an aside, deamination of 5-methyl-C creates thymine. Since C is the nucleotide typically methylated in eukaryotic cells, this is a significant problem because the cell cannot readily discern which base is “correct”.

18. Glycosylases for oxidized bases are typically bifunctional with intrinsic AP lyase activity Glycosylases not essential in mammals while APE1 is Some ends require cleaning by one of a variety of enzymes to be compatible for closing Single Nucleotide (SN) BER involves simple insertion and ligation Long Patch BER utilizes enzymes involved in DNA replication Typically 2-8 nucleotides are added After initial glycosylase cleavage and removal of modified base the AP site is repaired by differing mechanisms depending on the nature of the ends remaining. Blocked termini can arise when, for example, damage is ongoing while repair is taking place, or the repair is to an AP site that arose from ROS exposure rather than as a result of initial processing by glycosylases. Sites are resolved by two different processes which are essentially short and long patch repair. The long patch repair takes advantage of DNA replication machinery. The polymerase is unable to cleave off the blocking group so it is displaced and then cut away by the 5’ flap endonuclease FEN-1 which normally functions to remove Okazaki fragment primers during DNA replication. Ligase 1 seals the nick. Short patch BER is essentially filling in of a single nucleotide by Pol Beta followed by ligation by ligase III.After initial glycosylase cleavage and removal of modified base the AP site is repaired by differing mechanisms depending on the nature of the ends remaining. Blocked termini can arise when, for example, damage is ongoing while repair is taking place, or the repair is to an AP site that arose from ROS exposure rather than as a result of initial processing by glycosylases. Sites are resolved by two different processes which are essentially short and long patch repair. The long patch repair takes advantage of DNA replication machinery. The polymerase is unable to cleave off the blocking group so it is displaced and then cut away by the 5’ flap endonuclease FEN-1 which normally functions to remove Okazaki fragment primers during DNA replication. Ligase 1 seals the nick. Short patch BER is essentially filling in of a single nucleotide by Pol Beta followed by ligation by ligase III.

19. Once again, a genetic disorder is associated with deficiency in a DNA repair disorder. Prominent features again involve neurologic features. This raises a question given that many of the cells likely to be affected are post-mitotic. Is DNA repair important in such cells?Once again, a genetic disorder is associated with deficiency in a DNA repair disorder. Prominent features again involve neurologic features. This raises a question given that many of the cells likely to be affected are post-mitotic. Is DNA repair important in such cells?

20. Glycosylases for oxidized bases are typically bifunctional with intrinsic AP lyase activity Glycosylases not essential in mammals while APE1 is Some ends require cleaning by one of a variety of enzymes to be compatible for closing Single Nucleotide (SN) BER involves simple insertion and ligation Long Patch BER utilizes enzymes involved in DNA replication Typically 2-8 nucleotides are added E. coli has more than one APE while mammals have only a single protein, APE1. APE1 is essential and knockouts die very early in embryogenesis. Cell lines with floxed APE1 have been developed and expression of Cre in these cell lines leads to rapid cell death via apoptosis. APE1 also has a function in transcriptional regulation and it is unclear which function is essential. E. coli has more than one APE while mammals have only a single protein, APE1. APE1 is essential and knockouts die very early in embryogenesis. Cell lines with floxed APE1 have been developed and expression of Cre in these cell lines leads to rapid cell death via apoptosis. APE1 also has a function in transcriptional regulation and it is unclear which function is essential.

21. Single strand break sensors:PARP1 and XRCC1 PARP1 recognizes single strand breaks and nicks. An oligo with a single nick on one strand will be protected by PARP1. It’s activation results in poly ADP ribosylation of a number of proteins. This is metabolically costly exercise whose function is not entirely clear but appears to be a cellular signal of the presence of damage. XRCC1 acts as a scaffold for BER proteins but also stimulates the activity of a number of them including APE1, Pol Beta, and Ligase III. This might suggest a primary role in short patch repair.PARP1 recognizes single strand breaks and nicks. An oligo with a single nick on one strand will be protected by PARP1. It’s activation results in poly ADP ribosylation of a number of proteins. This is metabolically costly exercise whose function is not entirely clear but appears to be a cellular signal of the presence of damage. XRCC1 acts as a scaffold for BER proteins but also stimulates the activity of a number of them including APE1, Pol Beta, and Ligase III. This might suggest a primary role in short patch repair.

22. The major mechanisms of repair for nucleotide damage in mammals are via excision Base Excision Repair (BER) Repair of oxidizes, alkylated or inappropriate bases as well as abasic sites Responsible for repair of the majority of DNA damage Nucleotide Excision Repair (NER) Mechanism of DNA excision and repair synthesis that corrects damage caused by agents that create bulky DNA adducts (e.g. thymine dimers) Most versatile of excision repair mechanisms Mismatch Repair (MMR) Repairs base-base mismatches and insertion/deletion mispairings arising during DNA replication and recombination The second mechanism of DNA repair we will address is Nucleotide Excision Repair (NER). It functions to remove bulky DNA adducts but is typically thought of as the repair pathway for UV damage such as thymine dimers. It can, however, also remove other kinds of damage including some of the types handled by BER.The second mechanism of DNA repair we will address is Nucleotide Excision Repair (NER). It functions to remove bulky DNA adducts but is typically thought of as the repair pathway for UV damage such as thymine dimers. It can, however, also remove other kinds of damage including some of the types handled by BER.

23. NER in mammals is defined by it’s relationship with XP NER is the most complicated of DNA repair pathways involving perhaps 30 different proteins. At its core, are the proteins identified because of their disruption in XP which we discussed earlier. XP has multiple complementation groups which can be defined by cell fusion experiments and testing for UV sensitivity. These groups have proven to be representative of defects in different genes. However, as we shall see, there can be different mutations in the these genes that have very different phenotypic consequences. NER is the most complicated of DNA repair pathways involving perhaps 30 different proteins. At its core, are the proteins identified because of their disruption in XP which we discussed earlier. XP has multiple complementation groups which can be defined by cell fusion experiments and testing for UV sensitivity. These groups have proven to be representative of defects in different genes. However, as we shall see, there can be different mutations in the these genes that have very different phenotypic consequences.

24. There are two pathways of NER. One constantly surveys the genome for damage and carries our repair. This pathway was identified because it resulted in “unscheduled DNA synthesis”. This was work of Hanawalt. As indicated in the quotation, he later recognized a second form of NER that was focused on transcribed genes. This makes sense because some damage could presumably be tolerated if it occurred in non-coding regions, but damage to genes is likely to have a bigger impact. Note that NER, like BER, has distinct mechanisms for damage recognition and then that these pathways converge at the repair stage. Repair occurs through and ordered series of sequential recruitments and activities of proteins. Live cell imaging using different mutants has indicated the dependencies of downstream steps on the presence at the damaged site of upstream proteins.There are two pathways of NER. One constantly surveys the genome for damage and carries our repair. This pathway was identified because it resulted in “unscheduled DNA synthesis”. This was work of Hanawalt. As indicated in the quotation, he later recognized a second form of NER that was focused on transcribed genes. This makes sense because some damage could presumably be tolerated if it occurred in non-coding regions, but damage to genes is likely to have a bigger impact. Note that NER, like BER, has distinct mechanisms for damage recognition and then that these pathways converge at the repair stage. Repair occurs through and ordered series of sequential recruitments and activities of proteins. Live cell imaging using different mutants has indicated the dependencies of downstream steps on the presence at the damaged site of upstream proteins.

25. What does NER recognize?Both adduct and helical distortion required. Note no excision with mismatch alone or adduct alone.Note no excision with mismatch alone or adduct alone.

26. NER factors are freely diffusible in vivo Irradiate cells through a filter to generate localized regions of damage. See spots of immunreactivity to CPD antibodies, and subsequent localization of XPC. Determined epistatic relationship (e.g. get localization of XPB even in xpa cells, above). Time course indicates that factors are freely diffusible and not pre-assembled.Irradiate cells through a filter to generate localized regions of damage. See spots of immunreactivity to CPD antibodies, and subsequent localization of XPC. Determined epistatic relationship (e.g. get localization of XPB even in xpa cells, above). Time course indicates that factors are freely diffusible and not pre-assembled.

27. GG-NER is triggered by recognition of DNA distortion and bulky adducts Recognition of DNA damage triggers an ordered series of recruitment and activity at the damaged site. Resulting in incision on either side of the lesion, excision and gap-filling. Note that the damaged strand is always the one that is incised. Recognition of DNA damage triggers an ordered series of recruitment and activity at the damaged site. Resulting in incision on either side of the lesion, excision and gap-filling. Note that the damaged strand is always the one that is incised.

28. TC-NER is triggered by RNA polII stalling RNApolII interacts with CSB during elongation This interaction is stabilized by encounter with lesion Note CSB—Cockayne Syndrome B CSB is essential for recruitment of other factors Assembly of complex triggers chromatin remodeling Repair may require “backtracking of RNApolII and cleavage of protruding mRNA for transcription restart Transcription coupled repair occurs due to RNA polymerase stalling. Note that TC-NER involves Cockayne syndrome proteins—a second disorder associated with NER components.Transcription coupled repair occurs due to RNA polymerase stalling. Note that TC-NER involves Cockayne syndrome proteins—a second disorder associated with NER components.

29. The three key steps of NER after lesion recognition (which differs in GG-NER and TC-NER). Lesion demarcation occurs with RPA binding which is DNA single strand dependent. Incision by XPF and XPG. Gap filling involves familiar players seen in BER and also in DNA replication.The three key steps of NER after lesion recognition (which differs in GG-NER and TC-NER). Lesion demarcation occurs with RPA binding which is DNA single strand dependent. Incision by XPF and XPG. Gap filling involves familiar players seen in BER and also in DNA replication.

30. Xeroderma pigmentosum Autosomal recessive, first described in late 19th century. Defects in NER in XP cells were shown in late 1960s. Cell fusions examined for phenotypic correction in fusions with nuclei from cells derived from two different patients. XP results fundamentally from accumulation of unrepaired damage in skin due to sunlight exposure. However: Relationship between BER and NER and XP/CS: XPG (remember the 3’ incision endonuclease in NER) stimulates thymine glycol (Tg) DNA glycosylase-AP lyase (NTH1) repair. XPG specifically stimulates binding of NTH1 to its substrate, increasing both base excision and AP lyase activity. Stimulation requires the physical association of XPG with NTH1 but is independent of XPG nuclease activity. XPG/CS mutant cell lines with mutant (truncated) or no XPG have reduced Tg repair capacity, but cell lines with point mutations in XPG endonuclease do not have defects in Tg repair capacity. The suggestion is that CS (characterized by neurological and aging characteristics) in specific XPG patients is related to inefficient excision of oxidative damage. XPC stimulates thymine DNA glycosylase (TDG) activity. XPC appears to act similarly to Ape1 In that it competes with TDG for binding to the AP site product, thus increasing glycosylase turnover. XPC and Ape1 have an additive effect on TDG activity. Suggestion: XPC alone (or in combination with hHR23A,B) may participate in BER of G/T mismatches, thereby suppressing spontaneous mutagenesis that may be related to cancer in XP-C patients. Mouse models: targeted gene KOs mouse homologs, pretty much mimic the effects seen in human cells. Autosomal recessive, first described in late 19th century. Defects in NER in XP cells were shown in late 1960s. Cell fusions examined for phenotypic correction in fusions with nuclei from cells derived from two different patients. XP results fundamentally from accumulation of unrepaired damage in skin due to sunlight exposure. However: Relationship between BER and NER and XP/CS: XPG (remember the 3’ incision endonuclease in NER) stimulates thymine glycol (Tg) DNA glycosylase-AP lyase (NTH1) repair. XPG specifically stimulates binding of NTH1 to its substrate, increasing both base excision and AP lyase activity. Stimulation requires the physical association of XPG with NTH1 but is independent of XPG nuclease activity. XPG/CS mutant cell lines with mutant (truncated) or no XPG have reduced Tg repair capacity, but cell lines with point mutations in XPG endonuclease do not have defects in Tg repair capacity. The suggestion is that CS (characterized by neurological and aging characteristics) in specific XPG patients is related to inefficient excision of oxidative damage. XPC stimulates thymine DNA glycosylase (TDG) activity. XPC appears to act similarly to Ape1 In that it competes with TDG for binding to the AP site product, thus increasing glycosylase turnover. XPC and Ape1 have an additive effect on TDG activity. Suggestion: XPC alone (or in combination with hHR23A,B) may participate in BER of G/T mismatches, thereby suppressing spontaneous mutagenesis that may be related to cancer in XP-C patients. Mouse models: targeted gene KOs mouse homologs, pretty much mimic the effects seen in human cells.

31. Cockayne syndrome

33. Repair vs Transcriptional Roles for XP/CS Proteins Defects in common pathway give rise to both sets of symptoms depending on gene functions. Damage to whole genome (mostly nontranscribed) is processed by XPE, XPC and other NER factors as well as TLS Pol eta. Defects in these could result in genomic instability that result in malignant transformation. Damage to transcriptionally active genes causes tx arrest, and defects in TCR might not cause a high degree of genomic instability, but might enhance apoptosis, resulting in neuronal loss.Defects in common pathway give rise to both sets of symptoms depending on gene functions. Damage to whole genome (mostly nontranscribed) is processed by XPE, XPC and other NER factors as well as TLS Pol eta. Defects in these could result in genomic instability that result in malignant transformation. Damage to transcriptionally active genes causes tx arrest, and defects in TCR might not cause a high degree of genomic instability, but might enhance apoptosis, resulting in neuronal loss.